The Smad2/3 branch of the TGF2 signaling pathway is necessary to sustain pluripotency in human embryonic stem cells (hESCs) and human induced pluripotent cells (hiPSCs). Despite its significance, the molecular circuitry underlying pluripotency, as well as differentiation, remains poorly understood in both systems. Recent work, including our own, has shown that embryonic specific microRNAs (miRNAs) play crucial roles in modulating the fate decisions in early vertebrate development and HESCs by modulating the Smad2/3 pathway. This pathway acts as morphogen, transducing activin/nodal signals and eliciting different outcomes based on concentration and duration of signaling. While tremendous progress has been made in the resolution of the molecular mechanism underlying miRNA biochemistry and mechanism of action, their role in biological processes, specifically during early embryonic development and cell fate determination, also remains poorly understood. The two specific aims of this grant are to bridge these two areas in order to continue our efforts toward a more comprehensive understanding of both. Preliminary experiments presented here demonstrate that different levels of inhibition of ongoing Smad2/3 signaling (mimicking morphogen effects) specifically modulate expression of several miRNAs in hESCs. Based on these findings I propose to investigate the regulatory mechanisms and the function of these Smad2/3-regulated miRNAs during pluripotency and differentiation of hESCs and hiPSCs. Our first goal is to determine the regulatory threshold of Smad2/3 signaling required for the proper expression of each miRNA, followed by the discrimination between direct versus indirect, and transcriptional versus post-transcriptional mechanisms of regulation. Target analysis for each threshold-specific miRNA will address their molecular function. Secondly, we will perform gain and loss of miRNA function directly in hESCs and hiPSCs. Taken together, the successful accomplishment of the aims proposed in this study will contribute to a more in depth understanding of the role of threshold-specific Smad2/3 activity during hESC fate acquisition when observed from the miRNA perspective, and establish the biological function of these selectively expressed miRNA in the same context. Moreover, given the similarities between cancer cells and embryonic stem cells and the established role of both the TGF2 pathway and miRNAs in tumorigenesis, the discovery and the functional characterization of relevant Smad2/3-regulated miRNAs in hESCs and hiPSCs will have a direct impact in the cancer field. PUBLIC HEALTH RELEVANCE: In this study we propose to investigate the regulatory mechanisms and the function of these Smad2/3-regulated miRNAs during both pluripotency and differentiation of hESCs. By targeting and resolving the role of the Smad 2/3 branch of the TGF2 pathway and their regulation of miRNAs in the context of hESC differentiation, these studies contribute to our basic understanding of molecular processes involved in fate determination in hESCs. Moreover, as this pathway is also involved in cancer, the resolution of the aims of this study will also contribute to our basic understanding of the molecular basis of tumor formation. Thus, the results obtained from the successful accomplishment of the aims of this grant will have a significant impact on biomedical science and public health.